We are interested in (i) the molecular mechanisms by which stimulus- induced neuronal depolarization is converted into transcription-dependent changes in neuronal and behavioral properties, and (ii) the mechanisms by which neurotrophins specify neuronal differentiation of neuronal precursor cells. To study these mechanisms we are identifying and characterizing "neuron-restricted" immediate-early genes (IEGs) preferentially induced by depolarization, and genes that are induced by NGF, but not by other stimuli. We constructed a phage library prepared by subtracting cDNA from stimulated PC12 pheochromocytoma cells with cRNA from stimulated fibroblast and hepatoma cells. The library was differentially screened, to identify cDNAs for "neuron-restricted" immediate-early genes. One clone encodes the rat homologue of synaptotagmin 4 (Syt4), a recently identified member of the synaptotagmin family. Syt4 is induced in PC12 cells by depolarization, calcium ionophore, and secretogogues, but not by growth factors or tumor promoters. Syt4 is present in brain, but not in other organs. Syt4 is induced in the hippocampus and the piriform cortex following kainic acid induced seizures. In contrast, Syt1 is not inducible in PC12 cells or brain. We also identified secretogranin as a neuron- restricted IEG induced by depolarization, but not growth factors. In the next grant period we will compare the biochemical properties of Syt4 and Syt1 to determine (i) if Syt4 can, like Syt1, also form complexes with neurexin, (ii) if Syt4 is a vesicle protein, (iii) if Syt4 and Syt1 are present in overlapping or distinct vesicle populations, and (iii) if the rates of synthesis and degradation of Syt4 and Syt1 differ. We will determine whether altering Syt4 expression in PC12 cells modulates depolarization-induced fusion of synaptic vesicles with presynaptic plasma membranes and/or neurotransmitter release. We will also use genetic approaches to study Syt4. We will create mice with disruptions in the Syt4 gene, for electrophysiological and behavioral studies. We will map the marine Syt4 gene, to determine whether Syt4 might be a candidate gene for neurodegenerative or behavioral mutations. It is likely that additional depolarization-specific neuronal IEGs exist, but have not been identified. We will use Syt4 and secretogranin as guides to rescreen our subtracted library. We have modified the representational difference analysis (RDA) procedure to enrich for DNA sequences preferentially induced by NGF in PC12 cells, and identified collagenase as an NGF-inducible gene. We will use RDA to identify NGF-induced genes necessary for PC12 differentiation, and to identify additional depolarization-specific IEGs.